U.S. patent application number 09/183866 was filed with the patent office on 2001-07-05 for method for monitoring nucleic acid assays using synthetic internal controls with reversed nucleotide sequences.
Invention is credited to DUBOIS, DWIGHT B., WALKERPEACH, CINDY R..
Application Number | 20010006800 09/183866 |
Document ID | / |
Family ID | 26743951 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010006800 |
Kind Code |
A1 |
WALKERPEACH, CINDY R. ; et
al. |
July 5, 2001 |
METHOD FOR MONITORING NUCLEIC ACID ASSAYS USING SYNTHETIC INTERNAL
CONTROLS WITH REVERSED NUCLEOTIDE SEQUENCES
Abstract
The present invention relates to methods and compositions that
provide a positive control to identify inhibition during a signal
amplification reaction. The methods and compositions of the present
invention are designed to run in the same tube or assay environment
as the experimental or target sample and contain a copy of the
target sequence in an inverted form.
Inventors: |
WALKERPEACH, CINDY R.;
(AUSTIN, TX) ; DUBOIS, DWIGHT B.; (AUSTIN,
TX) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
26743951 |
Appl. No.: |
09/183866 |
Filed: |
October 30, 1998 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60063922 |
Oct 31, 1997 |
|
|
|
Current U.S.
Class: |
435/91.1 |
Current CPC
Class: |
C12Q 1/6851 20130101;
C12Q 1/6851 20130101; C12Q 2545/101 20130101 |
Class at
Publication: |
435/91.1 |
International
Class: |
C12N 015/10 |
Claims
We claim:
1. An internal control cassette for use in a polynucleotide
detection assay in which a target sequence is detected, said target
sequence having primer binding sites flanking an internal target
sequence, said cassette comprising primer binding sites flanking an
internal control sequence, wherein the internal control sequence
comprises said internal target sequence in a reversed
orientation.
2. The internal control cassette of claim 1, wherein the primer
binding sites flanking an internal control sequence are the same as
those flanking the internal target sequence.
3. The internal control cassette of claim 1, further comprising the
nucleic acid sequence of SEQ. ID. NO. 5.
4. The internal control cassette of claim 1, wherein the cassette
is a component of a plasmid.
5. A method for detecting signal amplification inhibition in an
assay comprising the steps of: co-amplifying a target sequence and
an internal control cassette, wherein the internal control cassette
comprises the target sequence in a reverse orientation.
6. The method of claim 5, wherein the assay is selected from the
group consisting of PCR, branched DNA (bDNA)-based signal
amplification assays, nucleic acid sequence based amplification
assays (NASBA), and transcription mediated amplification (TMA).
7. The method of claim 5, wherein the target sequence comprises DNA
or RNA.
8. The method of claim 7, wherein the target sequence is a nucleic
acid sequence from a virus selected from the group consisting of
HSV, HIV, HCV, CMV, and HPV.
9. The method of claim 8, wherein the target sequence comprises the
nucleic acid sequence of SEQ. ID. NO. 1.
10. The method of claim 9, wherein the internal control cassette
comprises the nucleic acid sequence of SEQ. ID. NO. 2.
11. The method of claim 5, further comprising the step of assaying
products generated by the co-amplification.
12. The method of claim 11, wherein the step of assaying products
is a primer binding assay comprising the steps of: determining the
extent of product binding to a capture probe specific for the
internal control cassette product.
13. The method of claim 12, wherein the capture probe consists of
the nucleic acid of SEQ ID NO. 6.
14. A method for detecting signal amplification inhibition in an
assay comprising the steps of: contacting one or more hybridization
probes with both a target sequence and an internal control cassette
in the same medium, wherein the internal control cassette comprises
the target sequence in a reverse orientation.
15. The method of claim 14, wherein the assay is a molecular beacon
assay.
16. The method of claim 15, wherein the internal control cassette
comprises the nucleic acid sequence of SEQ. ID. NO. 2.
17. The method of claim 15, wherein the hybridization probe
comprises the nucleic acid sequence of SEQ. ID. NO. 8.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application 60/063,922.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to the use of an internal
positive control containing an inverse sequence to detect
inhibition and to provide an internal quantitation standard in a
nucleic acid assay.
[0003] Modern nucleic acid assay techniques allow researchers and
clinicians to detect molecules of interest that are present in
extremely low concentration. These assays use probes to
specifically amplify by several orders of magnitude and detect the
amount of the molecule of interest. However, when used
diagnostically, falsely negative results arising from inhibition of
the assay reaction dramatically reduce the predictive value of the
assay. Thus there is a strong need for a method to control for
inhibition of the assay reaction.
[0004] The Polymerase Chain Reaction (PCR) is an example of such an
amplification technique for the detection of target molecules. With
PCR it is possible to test blood samples for minute quantities of
nucleic acid from pathogens, such as the human immunodeficiency
virus (HIV). The technique can also be used to detect a variety of
different infectious agents in a number of different clinical
settings such as testing blood or donor organs for infection.
Negative results may be unreliable given the susceptibility of
these techniques to non-specific inhibition by a variety of
compounds. Thus, there is a requirement for methods to
differentiate true negative results from false negative results
secondary to inhibition of the assay.
[0005] For the foregoing reasons, there is a need for an accurate
reproducible positive control to detect inhibition in the PCR
reaction. The method of detecting inhibition is further applicable
to other signal amplification assays.
SUMMARY OF THE INVENTION
[0006] The present invention relates to compositions and methods
that provide a positive control to identify inhibition during a
signal amplification reaction. The methods and compositions of the
present invention are designed to run in the same tube or assay
environment as the experimental or target sample and contain a copy
of the target sequence in an inverted form.
[0007] One embodiment of the present invention, provides for an
internal control cassette for use in a polynucleotide detection
assay in which a target sequence is detected. The target sequence
has primer binding sites flanking an internal target sequence. The
cassette comprises primer binding sites flanking an internal
control sequence, wherein the internal control sequence comprises
said internal target sequence in a reversed orientation.
[0008] In one aspect of this embodiment, the internal control
cassette further comprises one or more primer binding sites
adjacent to the internal control sequence. In another aspect, the
internal control cassette further comprises the nucleic acid
sequence of SEQ. ID. NO. 5. The internal control cassette may be a
component of a plasmid.
[0009] Another embodiment of the present invention contemplates a
method for detecting signal amplification inhibition in an assay
comprising the steps of: co-amplifying a target sequence and an
internal control cassette, wherein the internal control cassette
comprises the target sequence in a reverse orientation. Assays
contemplated for use with the present invention are selected from
the group consisting of PCR, real-time PCR, branched DNA
(bDNA)-based signal amplification assays, nucleic acid sequence
based amplification assays (NASBA), and transcription mediated
amplification (TMA).
[0010] The target sequences usable in the present invention include
any nucleic acid sequence that may be assayed with techniques known
in the art. In one aspect of this embodiment, the target sequence
comprises DNA or RNA. In another aspect, the target sequence is a
nucleic acid sequence from a virus selected from the group
consisting of HSV, HIV, HCV, CMV, and HPV. In another aspect, the
target sequence comprises the nucleic acid sequence of SEQ. ID. NO.
1.
[0011] Similarly, the internal control cassette sequences include
any sequence that may be a target sequence. For example, an
internal control cassette comprises the nucleic acid sequence of
SEQ. ID. NO. 2.
[0012] The methods of the present invention further comprise the
step of assaying products generated by the co-amplification
described above. The present invention further contemplates an
additional step of assaying products by a primer binding assay
comprising the step of determining the extent of product binding to
a capture probe specific for the internal control cassette product.
In one aspect of this embodiment, the capture probe consists of the
nucleic acid of SEQ ID NO. 6.
[0013] Another embodiment of the present invention contemplates a
method for detecting signal amplification inhibition in an assay
comprising the steps of contacting one or more hybridization probes
with both a target sequence and an internal control cassette in the
same medium, wherein the internal control cassette comprises the
target sequence in a reverse orientation. For example, the assay of
this embodiment is a molecular beacon assay. In another aspect, the
internal control cassette comprises the nucleic acid sequence of
SEQ. ID. NO. 2. In still another aspect, the hybridization probe
comprises the nucleic acid sequence of SEQ. ID. NO. 8.
DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 graphically depicts the HSV gB target and ICC
sequences.
[0015] FIG. 2 graphically depicts the amount of product produced in
a PCR reaction. The shaded bars represent HSV-related product and
the filled bars represent ICC-related product. The magnitude of the
bar are the optical density of the samples read at 450 nm.
[0016] FIG. 3 graphically depicts the amount of product produced in
a PCR reaction and the specificity of the signals produced therein.
The shaded bars represent HSV-related product and the filled bars
represent ICC-related product. The magnitude of the bar are the
optical density of the samples read at 450 nm.
[0017] FIG. 4 graphically depicts a comparison of samples assayed
using the Flowmetrix assay of Example 5 against the microtiter
based detection system of Example 3.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The present invention relates to methods and compositions
that provide a positive control to identify inhibition during a
signal amplification reaction. The methods and compositions of the
present invention are designed to run in the same tube or assay
environment as the experimental or target sample and contain a copy
of the target sequence in an inverted form.
[0019] The methods and compositions of the present invention offers
a number of advantages over other methods of controlling the
variables of signal amplification assays. One benefit of the
present invention's design relates to the possible presence of
inhibitors in the reaction mix. The methods described herein
provide the means to control for inhibition in the experimental
reaction.
[0020] Traditionally, a signal amplification assay is conducted
with an experimental sample and positive and negative controls run
in separate tubes, wells, or assay environments. The goal of these
assays is to examine the experimental sample for the presence or
absence of a target sequence. The positive control sample provides
a check to insure the functionality of the assay's reagents. The
negative control sample provides a means to determine the
background signal.
[0021] After the assay is run and the results are generated, one of
skill in the art will examine the experimental well for a signal.
The presence or absence of a signal indicates the presence or
absence of the target molecule. In the positive control sample, the
presence or absence of a signal indicates whether or not the
assay's reagents are functional. Thus, if there is no signal in the
experimental well but a signal in the control well, one of skill in
the art may conclude that there is no target sequence in the
experimental well, since the result from the control well indicates
that the assay's reagents are functional.
[0022] This conclusion will be accurate if the reason for the
absence of signal from the experimental well is that there is no
target sequence contained therein. If, on the other hand, there are
contaminants in the experimental sample that result in the
inhibition of the signal amplification reaction, then no signal
will be produced even though a target sequence is present in the
experiment sample. The positive control sample will produce a
signal, indicating that the reagents used in the assay are
functional. Under these conditions, one of ordinary skill in the
art could incorrectly interpret the results from signal
amplification assay as a negative result.
[0023] The methods and compositions of the present invention are
superior to traditional standards because, by combining
experimental and control fragments into one environment, one is
capable of detecting the presence of an inhibitor in the reaction
mixture. This capability is especially important in the clinical
setting. In a conventional prior art positive control system, when
a blood sample is tested for the presence of a particular target
molecule, such as a viral nucleic acids, the experimental and
control reactions are tested separately. A positive control sample
is run along side the experimental sample to insure that the
reagents used in the reaction are functioning properly. For
example, the experimental tube could contain a sample of blood, the
amplification primers, and the other reagents. In a separate tube a
positive control reaction is set up using a known target sequence,
appropriate primers, and a sample of the same reagents used in the
experimental tube. After the PCR reaction is complete, the results
are examined.
[0024] If a positive control for inhibition is included in the
experimental tube, (i.e., an internal positive control) then the
clinician can more confidently determine whether the target
molecule was actually present, or whether the results obtained were
merely a false negative. If no signal is obtained from the assay's
experimental well, then there was inhibition of the assay reaction.
If a signal is obtained from only the internal positive control but
not from the target molecule, then it is reasonable to conclude
that there was no target molecule present in the sample. A benefit
of the present invention is that it permits an investigator to
differentiate a true negative result from a false negative caused
by inhibition in the experimental reaction sample.
[0025] The present invention contemplates utility for use as an
internal inhibition control in a variety of signal amplification
assays. Examples of signal amplification assays include: the
polymerase chain reaction (PCR), variations of PCR, including
reverse transcriptase PCR, real-time PCR, branched DNA (bDNA)
assays, nucleic acid sequence based amplification assays (NASBA),
transcription mediated amplification (TMA), cytoflowmetric assays,
molecular beacon assays, hydridization reactions, and detection
assays.
[0026] The internal control cassette (ICC) may consist of any
target sequence that may be amplified by PCR or other nucleotide
amplification techniques. Sequences that are present in clinically
important disease states are particularly relevant to the present
invention. Examples provided for illustrative purposes include
sequences from viruses such as human immunodeficiency virus (HIV),
herpes simplex virus (HSV), hepatitis C virus (HCV), human
papilloma virus (HPV), and cytomegalovirus (CMV). Examples of
particular genes that may be used as target sequences include the
HSV gB gene and the HIV gp24 and gag genes.
[0027] As discussed above, the present invention provides a method
to control for inhibition of signal development in an experimental
well within a signal amplification assay. The ICC of the present
invention provides the template from which the internal control
signal is generated.
[0028] The ICC constructs of the present invention may be comprised
of a number of elements used to generate the control signal. The
ICC advantageously comprises a target sequence with an internal
segment in an inverted orientation, with respect to the target
sequence as it appears in nature, and one or more primer binding
sites. The segment of sequence inverted in the ICC preferably lies
immediately adjacent to or is flanked by the amplification primer
binding site or sites that are used to amplify the experimental
target sequence.
[0029] The entire construct may be a plasmid or some other
replicating construct. Alternatively, the ICC may be a linear
fragment of nucleic acid or polymerized amino acids. When the ICC
is a plasmid, the plasmid may be of bacterial origins.
[0030] A plasmid used to construct the ICC can advantageously
contain all of the necessary components found in any bacterial
plasmid used in the field of molecular biology. For example, an
origin of replication which would permit it to replicate within
host bacteria may be included. Also, a suitable plasmid may contain
a drug resistance marker, such as the ampicillin or tetracycline
resistant markers. Further, a suitable plasmid may contain a
multiple cloning site to facilitate the cloning in of the target
sequence to be used in the ICC. Other necessary or useful plasmid
components may also be included in a plasmid used to construct an
ICC, according to the judgment exercised by one of ordinary skilled
in the art.
[0031] The present invention also contemplates the use of an
internal control for RNA signal amplification assays. In a
preferred embodiment, a control plasmid for use in an RNA signal
amplification assay is prepared as described above. In another
preferred embodiment, an inducible promoter (such as lac) and a
sequence such as a poly(A) tail are cloned at the 5' and 3' ends of
the inverted target sequence (respectively) in the multiple cloning
site of the internal control plasmid. Using this plasmid it is
possible to produce RNA molecules (via in vitro transcription)
which contain the inverted capture sequence of a particular target
gene and as such would provide a suitable reagent with which to
control for inhibition of a RNA based amplification reaction.
[0032] Other methods of controlling for inhibition often use random
sequences of nucleotides as the target for the control reaction.
Nevertheless, use of random sequences does not accurately reflect
the biochemical limitations that come into play during the
amplification of a particular target sequence. These differences,
for example, in differences in nucleotide frequency or differences
in the overall chemical nature between a target sequence and a
control sequence may have a significant impact of the final
yield.
[0033] The present method differs from those methods of the prior
art in that it uses the same sequence as the target in the internal
control construct, although an internal segment is inverted. By
using an inverted sequence of a proposed target, as opposed to a
randomly generated control sequence, the present invention creates
a control sequence that shares many of the same biochemical
characteristics of the target sequence (e.g., reaction kinetics,
temperature of melting (T.sub.M), and nucleotide composition).
[0034] One shared feature is that the same primers may be used to
amplify both the control and target sequences. When the same primer
pair is used for both sequences, the hybridization or primer
annealing conditions for both the experimental and ICC control
sequences are the same. Thus, using the same primers and primer
binding sites for both sequences eliminates another variable which
might effect the signal produced from the control and target
sequences.
[0035] The choice of primers, their length and coding sequence are
a preference of one of ordinary skill in the art. For example, when
the primers are for use in a PCR reaction, the primers may be about
5 to 50 nucleotides long. Alternatively, each primer may be about
10 to 40 nucleotides long. In yet another alternative, each primer
may be 15 to 35 nucleotides in length.
[0036] Using the inverted control sequence of the present invention
also provides a number of quantitative similarities between the
sequences that improves the significance of the inhibition control.
This quantitative sequence similarity between target and control
ICC sequences provides a number of advantages over conventional
methods. For example, since the amplified sequences of the target
and control plasmids are of the same length and composed of the
same nucleotide bases, the reaction parameters for the two plasmids
are identical. Reaction parameters such as the T.sub.M, the length
of the sequence amplified, primer annealing or hybridization and
primer usage are all substantially the same for the experimental
and control sequences of the present invention. Given the
similarity in reaction parameters between the two sequences, the
yield of the co-amplification reactions should also be similar.
Thus the inverted sequence of the control plasmid provides an
extremely valid method for investigators to monitor for inhibition
during signal amplification reactions.
[0037] The present invention allows an investigator to control for
inhibition of a sequence amplification reaction with a control
sequence that has the same T.sub.M as the target sample. The
T.sub.M of interacting nucleic acid strands is determined by their
sequence. Here, the control and target sequences both have the same
composition of nucleotide base pairs arranged in a the same
sequence, albeit inverted. Therefore, the internal control and
target sequences have the same T.sub.M.
[0038] The importance of using a control sequence that binds assay
primers with the same T.sub.M as the target sample becomes clear
when the steps of the assay method are examined. For example,
during PCR subsequent rounds of denaturation, annealing and
polymerization are used to create a PCR product. During the
denaturation step, as the temperature of the rises, the forces
which hold the target strands together will be insufficient to keep
the molecule double stranded. When the template becomes single
stranded, it is available to bind the primers and the enzyme of the
PCR reaction.
[0039] Sequences that have higher T.sub.M.sub..sup.S may require
more heat to serve as efficient templates in the PCR reaction. This
comes from the fact that G, C, A, and T, each bind to each other
with either 2 or 3 hydrogen bonds. Thus, one utilizes a less
accurate control when the positive control sequence and its
corresponding primer or probe has a different Tm than that of the
experimental sample and its corresponding primer or probe.
Accordingly, one should avoid merely random sequences.
[0040] Another important feature of the present invention is the
length and composition of the control sequence. The length and
composition of the sequence amplified may effect the signal
obtained from the assay. During polymerization, the action of the
synthesizing enzyme traveling down the template strand is known as
processivity. The processivity of the enzyme may decrease as the
length of the target sequence increases. So, the longer the target
sequences, the more likely that it is that the PCR enzyme will fall
off the template before completing the synthesis of the replicated
strand. Premature termination of polymerization results in a
product that differs in length from the target sequence and that
difference could be misread as a negative result.
[0041] The positive control of the present invention eliminates
this problem. Since the amplified region of the positive control
plasmid is the same length as the target sequence, the rate of
premature termination should be the same for both sequences. As a
result, if there are apparent qualities of the target sequence
which cause the PCR enzyme to fall off, those same characteristics
should be present in the inverted control sequence and the PCR
enzyme should fall off that sequence as well.
[0042] Particular embodiments of the invention are discussed in
detail below. The following examples are for illustrative purposes
only and should not be interpreted as limitations of the claimed
invention. There are a variety of alternative techniques and
procedures available to those of skill in the art that would
similarly permit one to successfully perform the intended
invention.
EXAMPLE 1 Internal inhibition control plasmid construction
[0043] Construction of an internal control cassette (ICC) involved
creating a DNA fragment containing a portion of the HSV (herpes
simplex virus) gB gene (148 base pairs, nucleotide numbers
797-945), with the central 39 base pairs (nucleotide numbers
859-898) in the reverse orientation. (See FIG. 1). The gB gene was
discussed in Stuve, et al., "Structure and expression of the herpes
simplex virus type 2 glycoprotein gB gene." J. Virol.,
61(2):326-335 (1987) and Bzik, et al., "Nucleotide sequence
specifying the glycoprotein gene, gB of herpes simplex virus type
1." Virology 133:301-314 (1984)(herein incorporated by reference).
See also, Sutton, et al., Transgenic Research 1:228-236. (1992).
The total length of the ICC fragment was 160 base pairs which
includes the 148 base pair region and a unique restriction
endonuclease site on each terminus of the fragment (5'=EcoRI,
3'=Xhol) for cloning purposes.
[0044] De novo construction of the HSV-ICC fragment was performed
using PAGE purified oligodeoxynucleotides, a ligase chain reaction
developed for synthetic gene construction (modified from Sutton, et
al., 1992) and a thermostable ligase (Ampligase.TM., Epicentre
Technologies, Madison, Wisc.). After construction and cloning,
sequence verification was performed by dye-terminator sequence
chemistry (ABI 337).
[0045] A claiming plasmid pBluescript KS (Stratagene, San Diego,
Calif.) was chosen to carry the internal control sequence. Any
plasmid capable of replication in bacteria and suitable for
molecular biological manipulation may serve. The multiple cloning
region of the control plasmid was then cut with restriction
endonucleases which correspond to the sites on the de novo
constructed DNA fragment (which contained the central reversed
capture region). The restriction enzyme digestion created a linear
plasmid that accepted the exogenous nucleic acid sample that was
the internal control sequence. The digested control plasmid was
then isolated from the digestion reaction using standard techniques
known to those with ordinary skill in the art. Those techniques
include but are not limited to the use of glass beads, various
column matrices, gel isolation, etc.
[0046] The purified cleaved plasmid was then mixed with the
purified fragment and the DNA molecules were ligated together using
standard techniques known in the art. A DNA ligase was used to
close the phosphate backbone of the newly formed internal control
plasmid.
EXAMPLE 2 Internal inhibition control for the Polymerase Chain
Reaction (PCR)
[0047] Example 2 below discusses the inhibition detection
technology of the present invention for use with the polymerase
chain reaction. This method recites the use of a target nucleotide
sequence and a pair of primers that are complementary to that
sequence. The method discussed below also comprises the use of an
internal control plasmid in which the control sequence is the
inverted nucleotide sequence of the target sequence. The primers
used in the reaction are complementary to both the target sequence
and to the internal control construct. PCR was then performed on
the mixture.
[0048] In the PCR reaction, a target sequence is amplified by
several orders of magnitude using a template, a pair of primers and
a DNA polymerase enzyme. Typically, a control reaction is run
side-by-side with the experimental reaction to determine the
functionality of the reagents. PCR provides a powerful tool for
detecting small quantities of a target nucleic acid sequence in
solution. Nevertheless, the use of a traditional positive control
does not provide an investigator information as to whether or not
the polymerization reactions in the experimental tube has been
inhibited. The protocol described below includes the internal
inhibition control of the present invention that permits the
investigator to detect the presence of signal amplification
inhibition.
[0049] Once the templates, primers and reaction reagents are
collected and mixed, sufficient heat is applied to the sample to
denature the double stranded complex. Polymerization primers are
then permitted to anneal to the denatured template. The
primer-template complex of the PCR reaction is then bound by the
DNA polymerase enzyme. The Taq polymerase is an example of a PCR
suitable DNA polymerase. The DNA polymerase then proceeds to
synthesize a polynucleotide that is complementary to the target
strand. After a given period of time the enzyme is disassociated
from the template. The cycle is then repeated. Repetition of this
method permits the amplification of a given sequence as many as
4.times.10.sup.6 times in twenty-five cycles of the PCR reaction.
The PCR reaction is described in further detail in Mullis (1987)
U.S. Pat. No. 4,683,202, herein incorporated by reference.
[0050] A polymerase chain reaction is performed using the protocol
described below. This protocol is based on from Current Protocols
in Molecule Biology Volume 2, Chapter 15.1 (1995), which is
incorporated herein by reference. A PCR amplification buffer
concentrated 10 fold is prepared. The 10.times. PCR amplification
buffer contains: 500 mM KCl, 100 mM Tris-HCl, pH 9.0 (at 25.degree.
C.); 0.1% Triton X-100. The four nucleotide triphosphates (dNTPs)
are mixed for ease of application. For example, the 2.5 mM 4dNTP
mix is made by combining equal volumes of each dNTPs at a
concentration of 10 mM. These reagents are all commercially
available. Two primers, one in the forward and one in the reverse
orientation are designed based on standard principles known in the
art. These primers are diluted to a concentration of 20-50
pmol/.mu.l in H.sub.2O.
[0051] Template DNA is selected using standard parameters well
known in the art. In this Example, the template consisted of 1 pg
of viral genomic DNA/10 .mu.l. An inhibition control ICC is used in
combination with the 1 pg of genomic DNA a mixture containing
approximately 500 copies, bringing the final volume to 10
.mu.l.
[0052] An enzyme suitable for PCR, such as Taq DNA polymerase, is
used at a starting concentration of 5 U/.mu.l. Magnesium chloride
has been shown to effect PCR reactions, so three concentrations of
the salt were prepared: (L)15 mM, (M)30 mM and (H)45 mM MgCl.sub.2.
Sterile mineral oil was used to seal the reaction.
[0053] The PCR reactions are performed with the following volumes
of reagents (final concentrations): 10 .mu.l of the 10.times.PCR
amplification buffer, 1 .mu.l Primer 1 (0.5 .mu.M), 1 .mu.l Primer
2 (0.5 .mu.M), 10 .mu.l of Template DNA, 2 .mu.l of 10 mM 4dNTP
mix, 0.5 .mu.l of Taq polymerase (2.5 U), and H.sub.2O to 90 .mu.l.
The protocol requires that 90 .mu.l each of the master mix be
placed into three 0.2 ml tubes labeled L, M, and H. To each tube
was added 10 .mu.l of each corresponding concentration of
MgCl.sub.2 so that the final concentrations are 1.5, 3.0 and 4.5
mM, respectively.
[0054] A commercially available thermocycler is used to perform the
PCR reaction cycling of temperatures. The following steps listed
compose one PCR cycle. The reaction tubes are denatured for 1
minute at 94.degree. C. Next, the primers are annealed to the
template between 55 and 60.degree. C. depending on the T.sub.M of
the primers.
[0055] The primers are extended on the template at 72.degree. C.
for 1 to 3 minutes depending on the length of the target sequence.
The longer the sequence to be amplified, the longer the extension
time. Once the cycle is complete, the thermocycler returns to the
denature step. The reaction cycled for 25 to 30 times.
[0056] The results of the PCR reaction are assessed after the
reaction is complete. If the positive control shows PCR product by
detection methods well known in the art, the investigator will know
that the reagents used in the reaction are functional. If an
experimental signal is present, then the investigator knows that
the sample contains the molecule of interest. If, however, there is
no experimental signal, then the investigator needs to evaluate the
signal from the ICC. If a signal is present from the ICC but not
from the experimental sample, then the results are negative and the
investigator may conclude that there is no detectable target
sequence in the experimental sample. Alternatively, if there is no
signal from either the experimental sample or the ICC, then the
investigator may conclude that some inhibitory factor is present in
the experimental sample and that the negative results may or may
not indicate the presence of the target sequence in the
experimental sample.
EXAMPLE 3 Detection of Herpes Simplex Virus Type 1 (HSV-1) and Type
2 (HSV-2) in Cerebral Spinal Fluid by Qualitative Polymerase Chain
Reaction
[0057] This Example details procedures for using a PCR-based assay
for detecting HSV-1 and HSV-2 DNA in cerebral spinal fluid (CSF).
Detection of these viruses is an essential step in determining
whether patients are chronically infected with these specific
viruses.
[0058] The CSF used in this assay was obtained by spinal tap
according to techniques well known in the art. Upon collection of
the CSF, the sample was refrigerated at 1-4.degree. C. or frozen at
-20.degree. C. if storage lasted more than one week.
[0059] Samples obtained for amplification included CSF from two
patients Patient#21002 and Patient #28350, normal CSF (negative
control) and normal CSF spiked with cultured HSV as a positive
control. These samples were prepared and co-amplified with an ICC
to assay for the presence of HSV.
[0060] As described in the Example above, a master mix of reagents
was assembled for use in the PCR assay.
[0061] A 250 .mu.l sample of CSF was placed into a 2 ml
microcentrifuge tube and subjected to centrifugation at 1350
revolutions per minute (RPM) (1730 g) for 5 minutes. Two 100 .mu.l
samples of CSF were removed from the tube and pipetted into two
separate 1.5 ml conical microcentrifuge tubes. The next step was to
add 500 .mu.l phosphate buffered saline (PBS) into all experimental
and control tubes. This solution was vortexed well to mix the CSF
and PBS.
[0062] Control samples were prepared in 1.5 ml conical
microcentrifuge tubes to monitor the PCR reaction. The positive
control consisted of 100 .mu.L of 1 .mu.l stock HSV in 10 ml of
deionized water (ddH.sub.2O). The negative control consisted of 100
.mu.l of ddH.sub.2O. The marked patient samples and control tubes
were centrifuged for 1 hour at 21,000 RPM (39,444 g) at 4.degree.
C. At this point there were two samples per patient and and two
negative and positive controls.
[0063] After removing the supernatant from the spun samples, being
careful not to aspirate any precipitated matter. Approximately 10
.mu.l of supernatant remained in the tube after aspiration. Next 10
.mu.l of 0.025% BSA was added to the Lysis Buffer, which contained
0.4% (weight to volume) tergitol type NP-40; 1.25 mM DTT; and 4,000
copies of ICC/ml. In turn, 100 .mu.l of this lysing reagent was
added to each tube. The tubes were left to incubate at room
temperature for 10 minutes. After the incubation, 50 .mu.l of the
solution from each sample was placed into a separate PCR tube.
[0064] To that tube was added 50 .mu.l of the master mix. The
master mix contained the following reagents: PCR reaction buffer,
25 mM MgCl.sub.2, 10 mM dNTP, 20 mM dUTP, 100 .mu.M PCR Primer
gB.sub.1 (SEQ ID NO. 3), 100 .mu.M PCR Primer gB.sub.2 (SEQ ID NO.
4), 1 Unit/.mu.l of HK-UNG (Thermolabile Uracil N-Glycosylase;
Epicentre Technologies, Madison, Wisc.), 5 Units/.mu.l Taq, and
ddH.sub.2O. The primers were biotinylated to facilitate product
detection following the amplification reaction. After the addition
of the master mix, the tubes were placed into a thermocycler and
amplified. Table 1 below describes the steps of the program.
1TABLE 1 Thermocycler Program #CYCLES TIME AND TEMPERATURE 1 30
minutes at 37.degree. C. 1 3 minute at 95.degree. C. 5 95.degree.
C. for 45 seconds, 64.degree. C. for 45 seconds, 72.degree. C. for
45 seconds 30 95.degree. C. for 15 seconds, 64.degree. C. for 15
seconds, 72.degree. C. for 15 seconds
[0065] After the completion of the amplification, the samples were
removed from the thermocycler. Denature Solution consisting of 1.6%
NaOH, 1 mM EDTA and amaranth dye (Roche)(25 .mu.l) was added to
each sample followed by mixing. These samples were analyzed for the
presence of replicated materials and are discussed below.
EXAMPLE 3 Assay for Determining the Presence of Replication
Products
[0066] A microtiter plate based assay was used to determine the
presence or absence of amplification products. This assay utilized
a DNA capture probe to bind to assay the products of the PCR
reaction described in Example 2. This microtiter assay may also be
used to assay amplification products produced by amplification
reactions other than PCR.
[0067] Capture probes, such as the HSV gB probe (SEQ. ID. NO. 5) or
the ICC probe (SEQ. ID. NO. 6) were coupled to an amino group at
the 5' ends of the capture probes via a six carbon linker. The
probes were synthesized with the linker already attached using
standard phosphoamidite chemistry, which is well known in the art.
The HSV gB probe was an antisense probe with a sequence that
corresponded to nucleotide positions 1803 to 1841 of the gB gene of
both HSV-1 and HSV-2. The ICC capture probe was specific for the
ICC PCR product. (See FIG. 1).
[0068] The labeled capture probes were applied to the wells of high
binding flat bottom 1.times.8 strip well microtiter plates. (Coming
Costar). To the plate was added 100 .mu.l/well of amine modified
capture probe in probe binding buffer (50 mM Na2PO4, pH 8.5, 1 mM
EDTA) at a concentration of 25 pmol/well or greater. The plate was
then incubated overnight at 4.degree. C. The unbound probe was
removed from the plate by washing the plate three times with PBS.
Next, the plate was blocked by adding 200 .mu.l of 3% BSA in the
probe binding buffer. This was incubated for 30 minutes at
37.degree. C. at which time the solution was decanted.
[0069] The amplified specimen samples, 50 .mu.l per reaction tube,
were the transferred into each well of the prepared plate and mixed
5 times. The plate incubated for 1 hour at 37.degree. C. Following
the incubation the plate was washed five times using 1.times.wash
solution with an automated tray washer. The 1.times. wash solution
consisted of 10 mM phoshate buffer (pH 7.2), 150 mM NaCl, 1 mM
EDTA, 0.5% PROCLIN 300. To the washed plates was added 100
.mu.l/well of conjugate solution. The conjugate solution consisted
of: 25 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1.25 .mu.g/ml
Streptavidin-horseradish peroxidase, and 0.1% (v/v) Tween-20. The
plate was incubated for 15 minutes at 37.degree. C. following
addition of the conjugate solution. Following this incubation, the
plate was again washed 5 times with 1.times. wash solution using an
automated plate washer. To the washed plates was added 100 .mu.l of
substrate solution that consisted of 51.4 mM Na.sub.2HPO4, 24.3 mM
Citric Acid, 1 mg/ml 3,3', 5,5'-Tetramethylbenzidine
Dihydrochloride, and 40% (v/v) N.N-Dimethylformamide. Following
addition of the substrate, the plate was covered to exclude light,
and was incubated at room temperature for 5 minutes. After the
incubation period, 100 .mu.l of stop solution was added to each
well. The plate was then read at an optical density of 450 nm and
the results are shown in FIG. 2.
[0070] FIG. 2 shows the enzymatic activity detected from the
samples prepared in Example 2. Patient #21002 is clearly positive
for HSV while Patient#28350 is negative for HSV viral DNA.
Additionally, the controls indicate that there were no inhibitory
substances in the PCR reaction mix, so that the absence of an HSV
signal may be confidently interpreted as the lack of HSV target
sequence, rather than a false negative. This conclusion was
supported by the strong signal produced by the ICC sequence in all
of the samples tested.
EXAMPLE 4 Specificity of HSV and ICC Capture Probes
[0071] To address the possibility of cross reactivity between the
HSV and ICC capture probes, genomic HSV DNA or purified ICC plasmid
DNA was amplified along with a water based negative control using
the biotinylated primers (SEQ. ID. NOS. 3 and 4) and reaction
conditions discussed above in Example 2. At the conclusion of the
PCR reaction, the samples containing the HSV amplicon (SEQ. ID. NO.
1) or the ICC amplicon (SEQ. ID. NO. 2) or no amnplicon (negative
control) were individually alkali denatured (denaturing reagent)
and aliquoted into microtiter plate wells and allowed to hybridize
in neutralizing buffer. The assay was performed as described in
Example 3.
[0072] The wells contained a solid-phase bound oligonucleotide
sequence specific probe specific for either HSV (SEQ. ID. NO. 5) or
the ICC amplicon (SEQ. ID. NO. 6). After the hydridization and
washing steps of the assay protocol, an avidin-horseradish
peroxidase (AV-HRP) reagent was added to the wells. The AV-HRP
bound to the biotin-labeled PCR products that were in turn bound to
the plate via their interaction with the capture probes. The bound
AV-HRP conjugate present in each well was detected by a reaction
with peroxide and tetramethylbenzidine to form a colored product.
The optical density (OD.sub.450) was determined
spectrophotometrically. The results of this experiment are shown in
FIG. 3.
[0073] The data in FIG. 3 show that the HSV PCR product bound
specifically to the HSV capture probe containing wells, while the
ICC product only bound to the ICC capture probe containing wells.
These results show the specificity of the various capture probes
for their target sequences.
Alternative Probe Detection Assays
[0074] One aspect of the present invention contemplates a probe
specific for the internal inhibition control plasmid bound to a
fluorescently labeled microsphere. For example, probes specific for
target sequence and ICC PCR products, respectively, are used to
assay for the presence of herpes simplex virus (HSV) DNA in a
sample using the FlowMetrix.TM. cytometric microsphere technology
(Luminex Corp., DeSoto, Tex.).
EXAMPLE 5 Detection of PCR Products Using Flow Cytometric
Microsphere Technology
[0075] In this Example, two sequence specific oligonucleotide
probes, one for the HSV gB gene PCR product (SEQ ID NO 5) and one
for the ICC product (SEQ ID NO 6) were individually bound to two
different subsets of microspheres. The microsphere subsets were
distinguishable based on unique levels of incorporated orange and
red fluorescent dyes. These microsphere subsets were then mixed to
form a multiplex set. Each of the complementary probe target pairs
represented the internal sequence specific region within the HSV gB
gene and the ICC control plasmid.
[0076] Upon completion of the PCR reaction described in Example 2,
the amplification products are hybridized in a multiplex reaction
containing both of the labeled probes and the target microspheres.
Fluorescence from the probes is produced and detected by a flow
cytometer. (Smith et al., Clinical Chemistry 44:2054-2056 (1998);
van Huisden, et al., J. Histochem. 45:315-319 (1997)). If there are
inhibitory contaminants within the PCR reaction mixture, then no
signal will be seen from either the target sequence or the control
plasmid.
[0077] The presence of the control plasmid allows the investigator
to differentiate a true negative result due to the absence of the
target sequence from a negative result due to endogenous inhibition
of the PCR reaction. This Example illustrates the utility of this
invention, for without the presence of an internal control for
inhibition, an investigator would be unable to discern whether the
negative result indicated a lack of target or merely internal
amplification inhibition.
[0078] To illustrate this aspect of the present invention, a serial
dilution series of HSV and ICC PCR products, (in equimolar ratios)
was aliquoted. Each mixed dilution was detected by both the
microtiter capture plate assay described in Example 3 and by
FlowMetix flow cytometry. The data are shown in FIG. 4. Duplicate
samples were analyzed and averaged. Optical densities for the
microtiter plate data are on the x-axis while data from FlowMetrix
is on the y-axis. The ICC amplicon data is shown in FIG. 4A. HSV
amplicon detection data are in FIG. 4B. These results show a high
degree of correlation between the two detection methods.
[0079] Use of the ICC for amplification assay calibration and
quantification
[0080] In another embodiment of the present invention, the ICC may
be used as a means to calibrate or quantify the product of an
amplification reaction (signal or target). For example, the
addition of a known quantity of the ICC to an amplification
reaction would permit an investigator to quantitate the amount of
target sequence produced against the amount of ICC product
produced. The comparison would be especially meaningful since both
products are produced within the experimental sample tube of the
amplification reaction.
[0081] Quantification of a PCR Target Product by Comparison with an
ICC Standard
[0082] This Example provides a method to quantify the amount of
target PCR sequence produced during an amplification assay. The
comparison is made possible by knowing the starting concentration
of ICC and calculation of the PCR product over a known
amplification cycle profile. The ICC is constructed as discussed
above. A number of experimental reactions may be amplified
containing a number of different ICC starting concentrations. The
ICC products may be used as a standard curve with which to predict
the amount of starting target sequence. As in the previous
examples, the ICC and target templates are present in the same
experimental tube. The ICC template and primers may also be run in
a separate reaction tube to compare ICC product formation in the
presence and absence of target sample.
[0083] The PCR reaction is run as described in Examples 1 and 2.
The products of that reaction are analyzed as described in Example
3. ICC products produced in the experimental and control tubes are
compared and quantified. The amount of product produced by the ICC
amplifications is used to construct a standard curve. The amount of
target sequence product in the experimental wells is then compared
to the amount of ICC product produced. By comparing the amount of
product produced from the target sequence with that of the ICC
standards, the starting concentration of target sequences is
determined.
EXAMPLE 6
[0084] In this Example, a PCR reaction including a known quantity
of the ICC is run as described in Examples 1 and 2. A variety of
methods are known by one skilled in the art for the quantification
of plasmid DNA as well as PCR production quantification (e.g.,
absorbance at 260 nm). The amount of target PCR product can be
quantified by comparison to the amount of ICC PCR product produced
from a known amount of ICC added to the target amplification
reaction.
[0085] The internal control plasmid may also be used to perform
calibration of inhibitory factors found in patient samples.
[0086] An internal control for branched oligonucleotide signal
amplification
[0087] An embodiment of the present invention consists of a control
sequence wherein the entire internal sequence being reversed. In
this configuration, the present invention may be used as an
internal control for a branched oligonucleotide signal
amplification assay. The branched oligonucleotide signal
amplification assay uses a series of probes to bind and amplify a
target sequence. For example, human immunodeficiency virus type
(HIV-1) RNA was detected and quantified using a branched DNA signal
amplification by Pachl et al. (See Pachl et al., "Rapid and Precise
Quantification of HIV-1 RNA in Plasma Using a Branched DNA Signal
Amplification Assay," Journal of Acquired Immune Deficiency
Syndromes and Human Retrovirology, 8: 446-454 (1995); herein
incorporated by reference).
[0088] In the branched DNA assay (bDNA), the target is bound to a
well of a microtiter plate by oligonucleotide probes that bind to
and capture the target sequence. After binding, the bound
target-capture probe complexes are exposed to another
target-capture probe that is also capable of binding a branched DNA
molecule as well as the target molecule. An enzyme labeled branched
DNA probe is then added to the mixture and exposed to the substrate
of the enzyme label. The signal obtained from this enzymatic
reaction can be used to quantify the amount of target originally
captured by the assay.
[0089] The present invention provides a method to control for
internal inhibition of the bDNA assay. By using an internal control
composed of an inverted sequence of the target molecule, inhibition
of primer binding can be controlled. In one embodiment, an inverted
control plasmid is constructed and the RNA produced from this
plasmid is introduced along with the target sample into the
microtitre well. Unlike in the previous embodiments, the same
capture probes would not be used for both the control and the
target sequences. However, due to the similarity of the sequences,
the make up and binding of the control probes to the control
sequences would accurately reflect the binding that occurs on the
target molecule.
[0090] In a manner similar to the PCR example discussed above, the
presence of an internal control of inhibition would permit an
investigator to more accurately arrive at result indicating a
negative or positive result.
EXAMPLE 7
[0091] Blood is obtained by routine phlebotomy techniques and
tested for the presence of HIV-1 RNA using a branched DNA signal
amplification assay. Heparin containing blood collection tubes
should not be used as the presence of heparin appears to negatively
effect the concentration HIV-1 RNA in the plasma. (See Pachl et
al.). Plasma is obtained from the blood samples using
centrifugation at 800 g for 10 minutes. The plasma is stored at
less that -70.degree. C.
[0092] Plasma specimens are treated by the addition of 50 .mu.l of
a 0.1% red polystyrene 0.5 .mu.m bead suspension (Bangs
Laboratories, Carmel, Ind.) in 10 mM Tris-HCl pH 8.0, 1 mM EDTA to
each plasma containing tube. The samples are centrifuged for 1 hour
at 23,500 g at 2-8.degree. C. The supernatant is then removed and
the viral pellets are extracted for use in the branched DNA
assay.
[0093] The virus pellets are extracted using 220 .mu.l of Specimen
Working Reagent [400 mM LiCl, 100 mM HEPES pH 7.5, 8 mM EDTA, 1%
lithium lauryl sulfate, 12 .mu.g/ml sonicated salmon sperm DNA,
0.04% Na azide, 0.04% Proclin 200 (Supelco, Bellefonte, Pa.,
U.S.A.), 2.2 mg/ml proteinase K, 0.375 pmole/ml of each HIV-1
target probe or control probe to mediate capture, 1.25 pmole/ml of
each HIV-1 or control probe to bind amplifier]. The ICC control
plasmid may be added to the sample virus pellet before extract or
after the extraction procedure is completed. The ICC and the
control primers may be kept separated from the target sample and
used in a separate well of the assay plate discussed below.
[0094] The mixture of virus pellet is then vortexed, incubated at
53.degree. C. for 20 minutes to extract the viral RNA, vortexed
again, and centrifuged at 23,500 g for 15 minutes to clarify the
pellet extract. The clarified extract (200 .mu.l) is then added to
the assay wells of a 96-well assay plate which is coated with
either HIV-1 capture probes or control probes or a mixture of the
two. Other standards in addition to the ICC inhibition control may
also be used.
[0095] The assay plate is then incubated at 53.degree. C. to permit
the binding of the target or ICC control molecules to their
respective probes. The wells are then allowed to cool to room
temperature for 10 minutes and are then washed twice with Wash A, a
standard saline citrate (SSC)-0.1% SDS buffer. An amplification
buffer containing 2.0 pmole/ml bDNA amplifier in Amplifier Diluent
(50% horse serum, 1.3% SDS, 6mM Tris-HCl pH 8.0, 5.times.SSC), is
added along with 0.5 mg/ml proteinase K and is incubated at
65.degree. C. for 2 hours. This incubation is followed by the
addition of 1 mM phenylmethylsulfonyl fluoride (PMSF) to inactivate
the proteinase K, and 0.05% each Na azide and Proclin 300.
[0096] The wells are then sealed and incubated at 53.degree. C. for
30 minutes in order to hybridize the bDNA amplifier molecules to
the target-probe or control-probe complexes on the microwell
surface. The wells were subsequently cooled and washed as above,
followed by the addition of 50 .mu.l of HIV Label Working Reagent
(4 pmole/ml alkaline phosphatase-labeled probe in Amplifier
Diluent). The wells are then sealed and incubated at 53.degree. C.
for 15 minutes to hybridize the alkaline phosphatase probe to the
immobilized bDNA amplifier molecules. The wells are then cooled as
above and washed twice with Wash A followed by three washes with
Wash B (0.1.times. SSC). A 50 .mu.l volume of chemiluminescent
substrate, an enzyme triggerable dioxetane substrate for alkaline
phosphate (Lumiphos 530, Lumigen, Detroit, Mich., U.S.A.) is added
to each well and incubated at 37.degree. C. for 30 minutes. Light
emission is then measured in a luminometer.
[0097] An internal control for RNA amplification reactions
(RT-PCR)
[0098] In another embodiment, the positive control is a RNA
molecule. When an RNA molecule is the target of a signal
amplification assay, a RNA molecule should be used as a positive
inhibition control. RT-PCR refers to the use of reverse
transcriptase in the PCR reaction. Since RNA molecules are more
labile to degradation than double stranded DNA, it is appropriate
to control for degradation using the same type of nucleic acid. In
a preferred embodiment, a PCR based RNA amplification assay is
used.
EXAMPLE 8
[0099] The following protocol is based on Current Protocols in
Molecule Biology Volume 2, Chapter 15.4 (1995), which is
incorporated herein by reference.
[0100] A target RNA molecule is used as a template in the PCR
reaction. A sample of RNA is obtained through standard methods
known in the art. The RNA used in the signal amplification reaction
may be poly(A)+ RNA, or total RNA may be used. Alternatively
cytoplasmic RNA may be used. The type of RNA used in the reaction
will determine the type of control RNA used. The control RNA may
also be added directly to the sample containing the target RNA for
testing purposes.
[0101] A solution is prepared of 2 .mu.g of RNA, 25 ng (3 pmol)
cDNA primer, and sufficient H.sub.2O to bring the volume to 90
.mu.l. After mixing, 10 .mu.l of 3 M sodium acetate, pH 5.5 and 200
.mu.l of 100% are added. This solution is mixed and allowed to
precipitate overnight at -20.degree. C. or for 15 minutes at
-70.degree. C. The sample is then centrifuged for 15 minutes at
high speed at 4.degree. C. The resulting pellet is saved and the
supernatant is discarded. The pellet is washed with 70% ethanol and
centrifuged for 5 minutes at high speed, room temperature. The
supernatant is discarded. The pellet is dried briefly in a
desiccator.
[0102] The following ingredients are added to the RNA pellet: 12
.mu.l H.sub.2O, 4 .mu.l 400 mM Tris-HCl, pH 8.3, and 4 .mu.l 400 mM
KCl. The solution is heated to 90.degree. C. and then cooled slowly
to 67.degree. C. The sample is briefly microfuged to collect any
condensate that may have formed and incubated for 3 hours at
52.degree. C. Again, the sample is briefly microfuged to collect
any condensate.
[0103] A complementary or cDNA molecule is synthesized in the next
step. Twenty-nine microliters of reverse transcriptase buffer (50
mM Tris-HCl, pH 8.2, 5 mM MgCl2, 5 mM DTT, 50 mM KCl, 50 .mu.g/ml
BSA) and 0.5 .mu.l (16U) avian myeloblastosis virus (AMV) reverse
transcriptase are combined. These reagents are mixed and incubated
for 1 hour at 42.degree. C. One hundred fifty microliters of 10 mM
Tris-HCl/10 mM EDTA, pH 7.5 are combined and the solution is mixed
again. The solution is phenol extracted with 200 .mu.l buffered
phenol and vortexed. The sample is microcentrifuged for 5 minutes
at high speed, and the aqueous phase is saved. The mixture is
chloroform extracted with a solution of 24:1 chloroform:isoamyl
alcohol and vortexed. The sample is microcentrifuged for 5 minutes
at high speed, and the aqueous phase is retained. The solution is
precipitated with 20 .mu.l of 2M sodium acetate, pH 5.5, and 500
.mu.l of 100% ethanol overnight at -20.degree. C. or for 15 minutes
at -70.degree. C. The sample is microfuged for 15 minutes at high
speed for 4.degree. C. and the supernatant is discarded. The pellet
briefly dried and resuspended in 40 .mu.l of H.sub.2O. This
material is the template in the PCR reaction.
[0104] Following precipitation of the cDNA, the target and control
molecules are amplified using PCR. To a 5 .mu.l sample of cDNA 5
.mu.l of each amplification primer (20 .mu.M each) is added. To
that 4 .mu.l of 5 mM dNTP mix (see PCR protocol above), 10 .mu.l of
10.times. PCR amplification buffer and 70.5 .mu.l of H.sub.2O is
added and the reaction mixture is heated at 94.degree. C. The
sample is microcentrifuged and 2.5 U of Taq DNA polymerase is added
to the reaction mixture. The solution is overlaid with 100 .mu.l of
mineral oil before the reaction is run. Forty or more cycles in an
automated thermocycler are performed to amplify the target
molecules.
[0105] The reaction products are then assayed for the presence or
absence of control sequence as discussed in Example 3 or 5.
[0106] Enantiomeric and reversed amino acid sequence sequences used
for internal controls
[0107] Another aspect of the present invention contemplates the use
of reversed amino acid sequences and enantiomeric sequence as
controls for immunological assays. Enantiomers are compounds that
have the ability to rotate the plane of plane-polarized light as it
passes through a solution. Such compounds are asymmetric so that
they can exist in two different structural forms (D and L forms).
Each of the structural forms exist as mirror images of each other
and has the capability of rotating light in a particular direction.
Proteins are naturally occurring polymers of L-amino acids.
Synthetic enantiomers of naturally occurring proteins and smaller
peptide sequences can be readily chemically synthesized using
D-forms of amino acids. These peptides can then be used as controls
for immunoassays.
[0108] An important reagent in many immunoassays are antibodies.
Monoclonal or polyclonal antibodies are raised to a specific amino
acid sequence within the protein of interest. The techniques to
raise antibodies are well known in the art. For example, see
Antibodies: A Laboratory Manual, (Harlow and Lane, Eds.), Cold
Spring Harbor Laboratory (1988). The amino acid sequence of a
target protein, consisting of naturally occurring L-amino acids, is
determined using standard techniques known in the art. An
enantiomeric sequence, identical to the native epitope, only using
D-amino acids is synthesized and used to raise antibodies.
[0109] Chemical methods of peptide synthesis are well known in the
art. The use of tertiary-butyloxycarbonyl blocking groups (t-Boc
chemistry) or fluoromethoxy carbonyl blocking groups (Fmoc) are two
such methodologies. Once individual D-amino acids are synthesized
and fitted with blocking groups, a short D-peptide is synthesized.
In one embodiment solid-phase synthesis is used. Following
decoupling from the synthetic resin the D-peptides are purified
using high performance liquid chromatography (HPLC). The conditions
for such purification depend on the amino acids used to form the
D-peptides. The peptide sequences are confirmed through amino acid
sequencing techniques that are also well known in the art.
[0110] Following the synthesis of the D-peptides, an antibody would
be raised against it. Monoclonal antibody production is well known
in the art. Briefly, a target animal, preferably a mouse, would be
immunized with the D-peptides. After an appropriate number of
booster immunizations, the spleen of the immunized animal is
harvested and used to create hybridomas using techniques well known
in the field. Following hybridoma generation, individual colonies
are screened for antibody production. Those colonies that produce
active antibody can be expanded to produce large quantities of the
monoclonal antibody. (See Antibodies, A Laboratory Manual, Chapter
7, eds. Ed Harlow & David Lane, Cold Spring Harbor Laboratory
(1988), herein incorporated by reference).
[0111] In a preferred embodiment, the D-peptides and the monoclonal
antibodies generated against them are used as a control for
inhibition. Control D-peptides that are enantiomers to a known
target antigen are added to a sample in a detectable quantity. In a
preferred embodiment the control antigen is added in a range from 1
to 100 ng/well. Antibodies specific for both the target epitope and
the control D-peptides are added to the mixture. In a preferred
embodiment the two classes of antibodies are bound to two different
fluorescent bead populations so that the binding of control and
target antibodies may be determined. As with the other embodiments
of this invention, the absence of binding of the control antibody
to the control D-peptides would indicate inhibition of the
assay.
[0112] Immunoassays are well known in the art and involve the
detection an antibody or an antigen. For example, a well-known form
of immunoassay is the enzyme linked immunosorbent assay or ELISA
assay. In this immunoassay an antigen is bound to the bottom of an
assay plate and exposed to a sample of plasma that contains
antibodies. The assay plate is then washed and then exposed to an
anti-antibody antibody coupled to some signal-producing molecule,
like an enzyme. The assay plate is then screened for the presence
of a signal. The presence of a signal indicates antibody binding
and therefore the presence of antigen. Often, the level of signal
produced can be used to determine the quantity of antigen bound to
the bottom of the well.
[0113] Assays such as the ELISA are extremely useful for screening
samples for the presence of particular proteins. For example, if a
person has been exposed to HIV then it is likely that that person
will carry antibodies specific for certain HIV proteins. If that
person wishes to donate blood, then their blood may be screened for
antibodies against HIV that would suggest that the donated blood
was contaminated with HIV. On the other hand, a negative result
could be improperly interpreted as the absence of HIV exposure if
that negative result was due to inhibition of the assay. If some
molecule was inhibiting the assay mechanism, an investigator could
incorrectly conclude that the sample tested was free of HIV
antibodies. An internal control for inhibition would permit
investigators who monitor the blood supply to accurately
differentiate a true positive from a false positive test
result.
EXAMPLE 9
[0114] A blood sample is taken using standard techniques known in
the art and tested for the presence of the HIV protein, gp24
protein. Antibodies to an epitope of the gp24 protein as well as
its enantiomer are generated as discussed above. The blood sample
is processed for use in an ELISA assay.
[0115] An ELISA assay is performed to test for the presence or
absence of the gp24 protein according to protocols well known in
the art. A standard microtiter plate containing positive and
negative controls as well as experimental wells is assembled. To
control for inhibition, a detectable amount of gp24 enantiomer
epitope is included in the experimental wells of the assay. In one
experimental well, an antibody specific for the gp24 epitope is
used in the assay. In another experimental well, an antibody
specific for the gp24 enantiomer epitope is used. Reagents that
detect the presence of anti-gp24 or gp24 enantiomer antibodies are
then added to the assay. The results from the assay are then
analyzed.
[0116] The presence of a negative response from the anti-gp24
containing wells of the assay suggests that the tested blood lacks
detectable gp24. Nevertheless, the lack of signal from this well
may instead result from inhibition of binding by the anti-gp24
antibody. To determine whether inhibition is present in the
experimental well, the results from the experimental well assayed
with the anti-gp24 enantiomer antibody are examined. If a positive
signal is present in this well, then there was no inhibition, since
the anti-gp24 enantiomer antibody reacted with its target epitope.
On the other hand, if there is no signal from the anti-gp24
enantiomer antibody containing wells, then inhibition of the ELISA
assay may be present and the investigator may not conclude that the
assayed blood sample is free from gp24.
[0117] An internal control for Nucleic Acid Amplification Based
Amplification (NASBA) and Transcription Mediated Amplification
(TMA)
[0118] In another aspect of the present invention, the ICC may be
used as an internal inhibition control for a NASBA target
amplification assay. The NASBA and TMA technologies are isothermal
nucleic acid amplification assays that are virtually identical in
principle and practice. These isothermal nucleic acid amplification
assays use oligonucleotide probes, an RNA dependent polymerase and
a reverse transcriptase to amplify a target sequence. An isothermal
nucleic acid amplification assay, unlike PCR, is an isothermal
amplification method, thus it does not cycle between high and low
temperatures to facilitate target amplification. For example, the
amplification of human immunodeficiency virus type (HIV-1) RNA was
described using a NASBA signal amplification (See Kievits et al.,
"NASBA TM isothermal enzymatic in vitro nucleic acid amplification
optimized for the diagnosis of HIV-1 infection," Journal of
Virological Methods, 35: 273-286 (1991); herein incorporated by
reference).
[0119] In a NASBA assay, for example, the target RNA molecule is
placed into solution with oligonucleotide primers and two
polymerases. The temperature of the sample is raised to prepare the
target molecules for amplification. The temperature is then lowered
to permit the primer bind to the template. After binding the
target, a DNA molecule complementary to the target sequence is
synthesized using a reverse transcriptase, such as the AMV-reverse
transcriptase. Subsequent to this synthesis, the RNA-DNA hybrid
molecule is digested with RNase H, removing the RNA strand. A
complementary strand to the single stranded DNA target sequence is
synthesized through another round of DNA polymerization using a
reverse transcriptase enzyme.
[0120] Following the creation of this double stranded DNA molecule,
single stranded RNA is synthesized from the double stranded DNA
template with T7 RNA polymerase. This step amplifies the original
target sequence 100 to 1000-fold. Simultaneously, new double
stranded DNA templates are being synthesized from the replicated
single stranded RNA templates produced in the first round of
amplification. From this series of reactions, the target sequence
is amplified. The products of this reaction can be assayed to
determine the presence or absence of a target molecule.
Quantitation of the product created by the reaction is possible
using the methods described herein. Also, with the inclusion of the
ICC sequence of the present invention, a clinician using NASBA to
assay for the presence or absence of a target molecule could
accurately determine if inhibition of the reaction occurred,
resulting in a possibly false negative response.
EXAMPLE 10
[0121] Blood is obtained by routine phlebotomy techniques and
tested for the presence of HIV-1 RNA using a NASBA signal
amplification assay. A nucleic acid sample is isolated from the
plasma using generally known methods. (See Boom et al., "A rapid
and simple method for purification of nucleic acids," J. Clin.
Microbiol. 28, 495-503 (1990); herein incorporated by reference).
The isolated nucleic acid is resuspended in water and stored at
-70.degree. C.
[0122] Two microliters of isolated nucleic acid solution are mixed
with 23 .mu.l of a reaction mixture containing (at a final
concentration in a 25 .mu.l reaction mixture): 40 mM Tris, pH 8.5,
12 mM MgCl.sub.2, 42 mM KCl, 15% v/v DMSO, 1 mM each of dNTP, 2 mM
each NTP, 0.2 .mu.M Primer 1, 0.2 .mu.M Primer 2, 2 .mu.l of ICC
internal control RNA in a known quantity at a sufficiently high
concentration to provide a signal from the NASBA assay.
[0123] The sample is incubated at 65.degree. C. for 5 minutes,
destabilizing any secondary structures in the nucleic acid target.
The mixture is cooled to 41.degree. C. and the primers are thus
annealed to the template. The amplification reaction is started by
adding 2 .mu.l enzyme mixture (0.1 .mu.g/.mu.L BSA, 0.1 units RNase
H, 40 units T7 RNA polymerase and 8 units AMV-reverse
transcriptase). The reaction mixture is incubated at 41.degree. C.
for 90 minutes. The reaction mixture is then assayed for target and
control signal amplification. The absence of a signal from the ICC
control signal indicates inhibition of the signal amplification
reaction.
EXAMPLE 11
[0124] Transcription-Mediated Amplification (TMA) Assay
[0125] A blood sample is tested for the presence of the HSV gB gene
transcript according to the TMA protocol described in Stary, A., et
al., "Performance of transcription-mediated amplification and
ligase chain reaction assays for detection of chlamydial infection
in urogenital samples obtained by invasive and noninvasive
methods." J Clin Microbiol. 36(9):2666-70 (1998). A negative
response from an experimental sample containing the ICC control
indicates the presence of inhibtion.
Molecular Beacons
[0126] The term molecular beacons relates to an assay system that
utilizes probes that fluoresce upon hybridization with a target
sequence. Molecular beacons are discussed in Tyagi & Kramer,
"Molecular Beacons: Probes that Fluoresce upon Hybridization"
Nature Biotechnology, 14:303-308 (1996) and in Giesenfor, et al.,
"Molecular beacons: a new approach for semiautomated mutation
analysis," Clinical Chemistry 44:482-486 (1998). The principle of
this assay system involves a probe that consists of a stem-and-loop
structure. The loop portion of the molecule is a probe sequence
complementary to a predetermined target sequence. The stem is
formed by annealing on either side of the probe sequence two
complementary arm sequences that are unrelated to the target
sequence. A fluorophore is attached to the end of one arm and a
quenching moiety is attached to the end of the other arm. The stem
keeps these two moieties in close proximity to each other and
quenches the signal from the fluorophore. Once the molecular beacon
binds to its target, the fluorophore is separated from the
quencher, permitting a signal to be generated from the probe. Thus,
the probe undergoes a spontaneous conformational change that forces
the arm sequences apart, thereby moving the fluorophore and the
quencher away from each other and resulting in the generation of
fluorescence.
[0127] As with any assay system, the production of a signal
provides a basis to conclude that the target sequence is present in
the experimental sample. The converse does not necessarily hold,
however, since the absence of a signal in a hybridization assay may
result from amplification inhibition rather than the absence of
target from the assayed sample. The present invention may be used
as an internal inhibition control to determine whether the absence
of a signal is a legitimate negative result or merely a result of
inhibition.
EXAMPLE 12
[0128] A blood sample is prepared from an individual to be screened
for the presence of HSV gB. The blood sample is obtained and
prepared by methods well known in the art. Molecular probes
specific for the HSV gB (SEQ. I.D. NO. 7) and ICC (SEQ. I.D. NO. 8)
sequences are constructed according to the method of Tyagi and
Kramer.
[0129] One hundred and fifty .mu.l of a 170 nM solution of
molecular beacon probe SEQ ID NO 7 and 8 are separately dissolved
in 100 mM Tris-HCl (pH 8) containing 1 mM MgCl.sub.2 that is
maintained at 25.degree. C. The fluorescence of each probe solution
is monitored at 490 nm with time in an LS-5B spectrofluorometer
(Perkin Elmer), using 1 cm path length QS curvettes (Hellma) whose
temperature is controlled by a circulating water bath. There is no
change in fluorescence with time, so a sample containing 5-fold
molar excess of target sequence and ICC are added to curvettes
containing the probes. The level of fluorescence emitted is
recorded.
[0130] The temperature of the sample is gradually increased to
denature the probe and promote hybridization from 25.degree. C. to
75.degree. C. at a rate of 2.degree. C./minute. As the temperature
increases, the amount of fluorescence detected from the samples
containing the ICC and ICC specific probe increases. In the
experimental reaction mix, no signal is detected from the HSV gB
probe. Since there is signal from the ICC probe, there is no
inhibition of this experimental preparation.
EXAMPLE 13 Molecular Beacons and Real-Time PCR Analysis
[0131] A blood sample is prepared from an individual to be screened
for the presence of HSV gB using real-time PCR. This procedure
entails monitoring the generation of fluorescence during the
various PCR cycles using an ABI 7700 Sequence Detector
(Perkin-Elmer/Applied BioSystems). The blood sample is obtained and
prepared for use in PCR by methods well known in the art. Molecular
beacons specific for the HSV gB (SEQ. I.D. NO. 7) and ICC (SEQ.
I.D. NO. 8) sequences are constructed according to the method of
Tyagi and Kramer. The molecular beacons are labeled with different
fluorescent probes to emit different signals when bound.
Alternatively, the beacons may be labeled with the same probe and
to added to different reaction mixtures to monitor target sequence
synthesis and control sequence synthesis separately.
[0132] The PCR samples are assembled as discussed in the Examples
above. PCR primers, SEQ ID NOS 3 and 4 are used to amplify the
target and control sequences. The PCR reaction buffer is as
described above with the addition of a fluorescent dye at 60 nmol
final concentration (ROX, Perkin-Elmer). The molecular beacons are
added directly to the PCR mix. To the individual PCR reactions are
added blood samples or controls. To each of these is added the PCR
mix as well as a sample of ICC. The PCR tubes containing the
individual reactions are then subjected to thermocycling.
[0133] At 95.degree. C., the molecular beacons are denatured and
have a random coil structure, allowing full fluorescence. During
the decrease of the temperature in the PCR cycle, the formation of
hairpins occurs, which causes a drop in fluorescence. In samples
containing lacking the HSV target sequence the molecular beacons
fails to bind to their complementary sequences and there is no
increase in the fluorescence detected. However, in those
experimental reactions containing the molecular beacon specific for
the control sequence, molecular beacon binding to their
complementary sequences does occur and fluorescence increases.
[0134] From these results the investigator may reasonably conclude
that there is no signal inhibition and the negative result observed
reflects an absence of target sequence in the experimental PCR
reaction.
* * * * *